Detecting Properties of Protein Using Protein Binding Assay (Part 2 of 3)

Question:

How to use Protein Binding Assay to Detect Properties of Proteins?

The Protein Man Says:

There are a lot of biochemical, biophysical and theoretical methods that can be used to analyze protein-protein interactions. However, for the purpose of this article, we will stick with the biochemical methods, specifically the use ofprotein binding assays, in analyzing such interactions.

Types of Protein Binding Assays

Co-immunoprecipitation (co-IP) assays. Recognized as the gold standard for analyzing protein-protein interactions, co-IP assays are best used with endogenous proteins. In using this method, the protein of interest is isolated by using a specific antibody and the binding partners that interact with the protein are identified by Western blotting. However, please take note that co-IP assays are not suitable for use as a screening approach and can only be used to verify suspected interaction partners. In addition, false positive interactions may also result due to the non-specific binding of the antibody to the co-precipitated protein.

Pull-down assays. Basically, this is a common variation of the immunoprecipitation assay but instead of using antibodies to isolate the protein of interest, pull-down assays use a "bait" protein to achieve this purpose. This technique can be used as a discovery tool for screening binding protein partners, studying previously unknown protein-protein interactions and for investigating various cellular pathwaysin vitro. In addition, it can also be used as a confirmatory tool to establish previously suspected but not yet proven protein-protein interactions.

Electrophoretic mobility shift assay (EMSA). The EMSA is regarded as one of the most important techniques in analyzing the degree of affinity or specificity of protein-DNA interactions and for studying gene regulation. This technique is primarily based on the observationthat protein-DNA migrate more slowly as compared to free linear DNA molecules when subjected to agarose gel electrophoresis. It is for this very reason that EMSA is also known as gel shift assay. EMSA can be used to detect low abundance DNA binding proteins present in the lysate in vitro.

Aside from the three methods discussed in this article, there are still a lot of ways by which you can detect the binding properties of your protein of interest. We will be discussing some of these in our next post so please watch out for it.

Image By - Photokanok